L-lysine-producing corynebacteria and process for the preparation of lysine

ABSTRACT

The invention relates to L-lysine-producing strains of corynebacteria with enhanced pyc gene (pyruvate carboxylase gene), in which strains additional genes, chosen from the group consisting of the dapA gene (dihydrodipicolinate synthase gene), the lysC gene (aspartate kinase gene), the lysE gene (lysine export carrier gene) and the dapB gene (dihydrodipicolinate reductase gene), but especially the dapA gene, are enhanced and, in particular, over-expressed, and to a process for the preparation of L-lysine.

This application is a continuation-in-part of U.S. application Ser. No.09/353,608, filed Jul. 14, 1999, now abandoned which is incorporatedherein by reference in its entirely.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to L-lysine-producing strains of corynebacteriawith enhanced pyc gene (pyruvate carboxylase gene), in which strainsadditional genes, chosen from the group consisting of the dapA gene(dihydrodipicolinate synthase gene), the lysC gene (aspartate kinasegene), the lysE gene (lysine export carrier gene) and the dapB gene(dihydrodipicolinate reductase gene), but especially the dapA gene, areenhanced and, in particular, over-expressed, and to a process for thepreparation of L-lysine.

2. Background Information

L-Lysine is a commercially important L-amino acid which is usedespecially as a feed additive in animal nutrition. The demand of suchfeed additives has been steadily increasing in recent years.

L-Lysine is prepared by a fermentation process with L-lysine-producingstrains of corynebacteria, especially Corynebacterium glutamicum.Because of the great importance of this product, attempts are constantlybeing made to improve the preparative process. Improvements to theprocess may relate to measures involving the fermentation technology,e.g. stirring and oxygen supply, or the composition of the nutrientmedia, e.g. the sugar concentration during fermentation, or the work-upto the product form, e.g. by ion exchange chromatography, or theintrinsic productivity characteristics of the microorganism itself.

The productivity characteristics of these microorganisms are improved byusing methods of mutagenesis, selection and mutant choice to givestrains which are resistant to antimetabolites, e.g.S-(2-aminoethyl)cysteine, or auxotrophic for amino acids, e.g.L-leucine, and produce L-lysine.

Methods of recombinant DNA technology have also been used for some yearsin order to improve L-lysine-producing strains of Corynebacteriumglutamicum by amplifying individual biosynthesis genes and studying theeffect on the L-lysine production.

Thus, EP-A-0 088 166 reports the increase in productivity, afteramplification, of a DNA fragment conferring resistance toaminoethylcysteine. EP-B-0 387 527 reports the increase in productivity,after amplification, of an lysC allele coding for a feedback-resistantaspartate kinase. EP-B-0 197 335 reports the increase in productivity,after amplification, of the dapA gene coding for dihydrodipicolinatesynthase. EP-A-0 219 027 reports the increase in productivity, afteramplification, of the asd gene coding for aspartate semialdehydedehydrogenase. Pisabarro et al. (Journal of Bacteriology 175(9),2743-2749 (1993)) describe the dapB gene coding for dihydrodipicolinatereductase.

The effect of the amplification of primary metabolism genes on theL-lysine production has also been studied. Thus EP-A-0 219 027 reportsthe increase in productivity, after amplification, of the aspC genecoding for aspartate aminotransferase. EP-B-0 143 195 and EP-B-0 358 940report the increase in productivity, after amplification, of the ppcgene coding for phosphoenolpyruvate carboxylase. DE-A-198 31 609 reportsthe increase in productivity, after amplification, of the pyc genecoding for pyruvate carboxylase. The anaplerotic reaction catalyzed bypyruvate carboxylase is of particular importance compared with thereaction catalyzed by phosphoenolpyruvate carboxylase. Thus, Wendisch etal. (FEMS Microbiology Letters 112, 269-274 (1993)) showed that thelysine production of the strain MH20-22B was not impaired by turning offthe ppc gene.

Finally, DE-A-195 48 222 describes that an increased activity of theL-lysine export carrier coded for by the lysE gene promotes lysineproduction.

In addition to these attempts to amplify an individual gene, attemptshave also been made to amplify two or more genes simultaneously, therebyimproving the L-lysine production in corynebacteria. Thus, DE-A-38 23451 reports the increase in productivity, after simultaneousamplification, of the asd gene and the dapA gene from Escherichia coli.DE-A-39 43 117 discloses the increase in productivity, aftersimultaneous amplification, of an lysC allele coding for afeedback-resistant aspartate kinase and of the dapA gene. EP-A-0 841 395particularly reports the increase in productivity, after simultaneousamplification, of an lysC allele coding for a feedback-resistantaspartate kinase and of the dapB gene; further improvements could beachieved by additional amplification of the dapB, lysA and ddh genes.EP-A-0 854 189 describes the increase in productivity, aftersimultaneous amplification, of an lysC allele coding for afeedback-resistant aspartate kinase and of the dapA, dapB, lysA and aspCgenes. EP-A-0 857 784 particularly reports the increase in productivity,after simultaneous amplification, of an lysC allele coding for afeedback-resistant aspartate kinase enzyme and the lysA gene; a furtherimprovement could be achieved by additional amplification of the ppcgene.

It is clear from the many processes described in the state of the art,that there is a need for the development of novel approaches and for theimprovement of existing processes for lysine production withcorynebacteria.

SUMMARY OF THE INVENTION Object of the Invention

It is an object of the invention to provide novel L-lysine-producingstrains of corynebacteria and processes for the preparation of L-lysine.

Description of the Invention

When L-lysine or lysine is mentioned in the following text, it should beunderstood as meaning not only the base but also the appropriate salts,e.g. lysine hydrochloride or lysine sulfate.

The invention provides L-lysine-producing strains of corynebacteria withenhanced pyc gene (pyruvate carboxylase gene), wherein additional genes,chosen from the group consisting of the dapA gene (dihydrodipicolinatesynthase gene), the lysC gene (aspartate kinase gene), the lysE gene(lysine export carrier gene) and the dapB gene (dihydrodipicolinatereductase gene), but especially the dapA gene, are enhanced and, inparticular, over-expressed.

A novel DNA sequence located upstream (5′ end) from the dapB gene hasalso been found, which carries the −35 region of the dapB promoter andis advantageous for the expression of the dapB gene. It is shown as SEQID No. 1.

A corresponding DNA capable of replication, with the nucleotide sequenceshown in SEQ ID No. 1, is also included in the invention.

The invention also provides the MC20 and MA16 mutations of the dapApromoter shown in SEQ ID No. 5 and SEQ ID No. 6, deposited in thestrains DSM12868 and DSM12867.

The invention furthermore provides L-lysine-producing strains ofcorynebacteria with amplified pyc gene, wherein additionally the dapAand dapB genes are simultaneously enhanced and, in particular,over-expressed.

The invention also provides L-lysine-producing strains of corynebacteriawith enhanced pyc gene, wherein additionally the dapA, dapB and lysEgenes are simultaneously enhanced and, in particular, over-expressed.

In this context the term “enhancement” describes the increase in theintracellular activity, in a microorganism, of one or more enzymes whichare coded for by the appropriate DNA, by increasing the copy number ofthe gene(s), using a strong promoter or using a gene coding for anappropriate enzyme with a high activity, and optionally combining thesemeasures.

In this context, “amplification” describes a specific procedure forachieving an enhancement whereby the number of DNA molecules carrying agene or genes, an allele or alleles, a regulatory signal or signals orany other genetic feature(s) is increased.

The invention also provides a process for the preparation of L-lysineusing the bacteria described above.

The microorganisms which the present invention provides can prepareL-lysine from glucose, sucrose, lactose, fructose, maltose, molasses,starch or cellulose or from glycerol and ethanol, especially fromglucose or sucrose. Said microorganisms are corynebacteria, especiallyof the genus Corynebacterium. The species Corynebacterium glutamicum maybe mentioned in particular in the genus Corynebacterium, being known tothose skilled in the art for its ability to produce amino acids. Thisspecies includes wild-type strains such as Corynebacterium glutamicumATCC13032, Brevibacterium flavum ATCC14067, Corynebacterium melassecolaATCC17965 and strains or mutants derived therefrom. Examples ofL-lysine-producing mutants of corynebacteria are:

-   -   Corynebacterium glutamicum FERM-P 1709    -   Brevibacterium flavum FERM-P 1708    -   Brevibacterium lactofermentum FERM-P 1712    -   Brevibacterium flavum FERM-P 6463    -   Brevibacterium flavum FERM-P 6464    -   Corynebacterium glutamicum DSM5714    -   Corynebacterium glutamicum DSM12866

It has now been found that an enhanced expression of the lysE gene inaddition to the pyc gene, or an additionally enhanced expression of anlysC allele coding for a feedback-resistant aspartate kinase, or anadditionally enhanced expression of the dapB gene and, in particular, anadditionally enhanced expression of the dapA gene, individually ortogether, further improve L-lysine production.

It has also been found that, for a given over-expression of the pycgene, the simultaneous, additionally enhanced expression of the dapA anddapB genes brings further advantages for L-lysine production.

Finally, it has been found that, for a given over-expression of the pycgene, the simultaneous, additionally enhanced expression of the dapA,dapB and lysE genes is extremely advantageous for L-lysine production.

An enhancement (over-expression) is achieved, e.g., by increasing thecopy number of the appropriate genes or mutating the promoter andregulatory region or the ribosome binding site located upstream from thestructural gene. Expression cassettes incorporated upstream from thestructural gene function in the same way. Inducible promotersadditionally make it possible to increase the expression in the courseof the formation of L-lysine by fermentation. Measures for prolongingthe life of the mRNA also improve the expression. Furthermore, theenzyme activity is also enhanced by preventing the degradation of theenzyme protein, the genes or gene constructs either being located inplasmids (shuttle vectors) of variable copy number or being integratedand amplified in the chromosome. Alternatively, it is also possible toachieve an over-expression of the genes in question by changing thecomposition of the media and the culture technique.

Those skilled in the art will find relevant instructions inter alia inMartin et al. (Bio/Technology 5, 137-146 (1987)), Guerrero et al. (Gene138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430(1988)), Eikmanns et al. (Gene 102, 93-98 (1991)), EP-0 472 869, U.S.Pat. No. 4,601,893, Schwarzer and Pühler (Bio/Technology 9, 84-87(1991)), Reinscheid et al. (Applied and Environmental Microbiology 60,126-132 (1994)), LaBarre et al. (Journal of Bacteriology 175, 1001-1007(1993)), WO 96/15246, Malumbres et al. (Gene 134, 15-24 (1993)),JP-A-10-229891, Jensen and Hammer (Biotechnology and Bioengineering 58,191-195 (1998)) or the handbook “Manual of Methods for GeneralBacteriology” of the American Society for Bacteriology (Washington D.C.,USA, 1981) and well-known textbooks on genetics and molecular biology.

The genes from Corynebacterium glutamicum used according to theinvention are described and can be isolated, prepared or synthesized byknown methods.

Methods of localized mutagenesis are described, inter alia, by Higuchiet al. (Nucleic Acids Research 16, 7351-7367 (1988)) or by Silver et al.in the handbook by Innis, Glefand and Sninsky (eds.) entitled “PCRStrategies” (Academic Press, London, UK, 1995).

The first step in isolating a gene of interest from C. glutamicum is toconstruct a gene library of this microorganism in, e.g., E. coli oroptionally also in C. glutamicum. The construction of gene libraries isdocumented in generally well-known textbooks and handbooks. Exampleswhich may be mentioned are the textbook by Winnacker entitled “FromGenes to Clones, Introduction to Gene Technology” (Verlag Chemie,Weinheim, Germany, 1990) or the handbook by Sambrook et al. entitled“Molecular Cloning, A Laboratory Manual” (Cold Spring Harbor LaboratoryPress, 1989). Bathe et al. (Molecular and General Genetics, 252: 255-265(1996)) describe a gene library of C. glutamicum ATCC13032 which wasconstructed using cosmid vector SuperCos I (Wahl et al., Proceedings ofthe National Academy of Sciences, USA 84, 2160-2164 (1987)) in E. coliK-12 NM554 (Raleigh et al., Nucleic Acids Research 16: 1563-1575(1988)). Börmann et al. (Molecular Microbiology 6 (3), 317-326) in turndescribe a gene library of C. glutamicum ATCC13032 using cosmid pHC79(Hohn and Collins, Gene 11, 291-298 (1980)). A gene library of C.glutamicum in E. coli can also be constructed using plasmids like pBR322(Bolivar, Life Sciences 25, 807-818 (1979)) or pUC19 (Norrander et al.,Gene 26: 101-106 (1983)). In the same way, it is also possible to useshuttle vectors such as pJC1 (Cremer et al., Molecular and GeneralGenetics 220, 478-480 (1990)) or pEC5 (Eikmanns et al., Gene 102, 93-98(1991)), which replicate in E. coli and C. glutamicum. Restriction-and/or recombination-defective strains are particularly suitable hosts,an example being the E. coli strain DH5αmcr, which has been described byGrant et al. (Proceedings of the National Academy of Sciences, USA 87,4645-4649 (1990)). Other examples are the restriction-defective C.glutamicum strains RM3 and RM4, which are described by Schäfer et al.(Applied and Environmental Microbiology 60(2), 756-759 (1994)).

The gene library is then transferred to an indicator strain bytransformation (Hanahan, Journal of Molecular Biology 166, 557-580(1983)) or electroporation (Tauch et al., FEMS Microbiological Letters,123: 343-347 (1994)). The characteristic feature of the indicator strainis that it possesses a mutation in the gene of interest which causes adetectable phenotype, e.g. an auxotrophy. The indicator strains ormutants are obtainable from publicized sources or strain collections,e.g. the Genetic Stock Center of Yale University (New Haven, Conn.,USA), or if necessary are specially prepared. An example of such anindicator strain which may be mentioned is the E. coli strain RDA8requiring mesodiaminopimelic acid (Richaud et al., C.R. Acad. Sci. ParisSer. III 293: 507-512 (1981)), which carries a mutation (dapA::Mu) inthe dapA gene.

After transformation of the indicator strain with a recombinant plasmidcarrying the gene of interest, and expression of the gene in question,the indicator strain becomes prototrophic in respect of the appropriatecharacteristic. If the cloned DNA fragment confers resistance, e.g. toan antimetabolite such as S-(2-aminoethyl)cysteine, the indicator straincarrying the recombinant plasmid can be identified by selection onappropriately supplemented nutrient media.

If the nucleotide sequence of the gene region of interest is known orobtainable from a data bank, the chromosomal DNA can be isolated byknown methods, e.g. as described by Eikmanns et al. (Microbiology 140,1817-1828 (1994)), and the gene in question can be synthesized by thepolymerase chain reaction (PCR) using suitable primers and cloned into asuitable plasmid vector, e.g. pCRIITOPO from Invitrogen (Groningen, TheNetherlands). A summary of PCR methodology can be found in the book byNewton and Graham entitled “PCR” (Spektrum Akademischer Verlag,Heidelberg, Germany, 1994).

Examples of publicly accessible data banks for nucleotide sequences arethat of the European Molecular Biologies Laboratories (EMBL, Heidelberg,Germany) or that of the National Center for Biotechnology Information(NCBI, Bethesda, Md., USA).

The isolation and cloning of the pyc gene from C. glutamicum ATCC13032are described in DE-A-198 31 609 and by Koffas et al. (AppliedMicrobiology and Biotechnology 50, 346-352 (1988)). The nucleotidesequence of the pyc gene is obtainable under accession number AF038548or Y09548.

The isolation and cloning of the lysE gene from C. glutamicum ATCC13032are described in DE-A-195 48 222. The nucleotide sequence of the lysEgene is obtainable under accession number X96471.

The isolation, cloning and sequencing of the dapA gene from variousstrains of C. glutamicum are described by Cremer et al. (Molecular andGeneral Genetics 220: 478-480 (1990)), by Pisabarro et al. (Journal ofBacteriology 175: 2743-2749 (1993)) and by Bonnassie et al. (NucleicAcids Research 18: 6421 (1990)). DE-A-39 43 117 reports theamplification of the dapA gene by means of plasmid pJC23. The nucleotidesequence of the dapA gene is obtainable under accession number X53993.

The isolation, cloning and sequencing of the dapB gene fromBrevibacterium lactofermentum are described by Pisabarro et al. (Journalof Bacteriology 175: 2743-2749 (1993)). The nucleotide sequence of thedapB gene is obtainable under accession number X67737.

The isolation, cloning and sequencing of the lysC gene and of lysCalleles coding for a feedback-resistant aspartate kinase are reported byseveral authors. Thus, Kalinowski et al. (Molecular and General Genetics224: 317-324 (1990)) report the lysC allele from the C. glutamicumstrain DM58-1. DE-A-39 43 117 reports the cloning of the lysC allelefrom the C. glutamicum strain MH20. Follettie et al. (Journal ofBacteriology 175: 4096-4103 (1993)) report the lysC allele from the C.flavum strain N13, which is called “ask” in said publication. Thenucleotide sequences of the lysC gene and of various lysC alleles areobtainable inter alia under accession numbers X57226 and E06826.

The genes obtained in this way can then be incorporated inter alia intoplasmid vectors, e.g. pJC1 (Cremer et al., Molecular and GeneralGenetics 220, 478-480 (1990)) or pEC5 (Eikmanns et al., Gene, 102, 93-98(1991)), individually or in suitable combinations, transferred todesired strains of corynebacteria, e.g. the strain MH20-22B (Schrumpf etal., Applied Microbiology and Biotechnology 37: 566-571 (1992)), bytransformation, e.g. as in Thierbach et al. (Applied Microbiology andBiotechnology 29, 356-362 (1988)), or by electroporation, e.g. as inDunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)), and expressed.The strain to be chosen can equally well be transformed with two plasmidvectors, each containing the gene or genes in question, therebyachieving the advantageous, simultaneously enhanced expression of two ormore genes in addition to the known enhancement of the pyc gene.

Examples of such strains are:

-   -   the strain MH20-22B/pJC23/pEC7pyc, in which the pyc and dapA        genes are simultaneously enhanced, or    -   the strain MH20-22B/pJC33/pEC7pyc, in which the pyc- and the        lysC(FBR) allele are simultaneously enhanced and, in particular,        over-expressed, or    -   the strain MH20-22B/pJC23/pEC7dapBpyc, in which the pyc, dapA        and dapB genes are simultaneously enhanced and, in particular,        over-expressed, or    -   the strain MH20-22B/pJC23/pEC7lysEdapBpyc, in which the pyc,        dapA, dapB and lysE genes are simultaneously enhanced and, in        particular, over-expressed.

The microorganisms prepared according to the invention can be cultivatedfor L-lysine production continuously or discontinuously by the batchprocess, the fed batch process or the repeated fed batch process. Asummary of known cultivation methods is provided in the textbook byChmiel (Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik(Bioprocess Technology 1. Introduction to Bioengineering) (GustavFischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas(Bioreaktoren und periphere Einrichtungen (Bioreactors and PeripheralEquipment) (Vieweg Verlag, Brunswick/Wiesbaden, 1994)).

The culture medium to be used must appropriately meet the demands of theparticular microorganisms. Descriptions of culture media for variousmicroorganisms can be found in the handbook “Manual of Methods forGeneral Bacteriology” of the American Society for Bacteriology(Washington D.C., USA, 1981). Carbon sources which can be used aresugars and carbohydrates, e.g. glucose, sucrose, lactose, fructose,maltose, molasses, starch and cellulose, oils and fats, e.g. soya oil,sunflower oil, groundnut oil and coconut fat, fatty acids, e.g. palmiticacid, stearic acid and linoleic acid, alcohols, e.g. glycerol andethanol, and organic acids, e.g. acetic acid. These substances can beused individually or as a mixture. Nitrogen sources which can be usedare organic nitrogen-containing compounds such as peptones, yeastextract, meat extract, malt extract, corn steep liquor, soybean flourand urea, or inorganic compounds such as ammonium sulfate, ammoniumchloride, ammonium phosphate, ammonium carbonate and ammonium nitrate.The nitrogen sources can be used individually or as a mixture.Phosphorus sources which can be used are potassium dihydrogenphosphateor dipotassium hydrogenphosphate or the corresponding sodium salts. Theculture medium must also contain metal salts, e.g. magnesium sulfate oriron sulfate, which are necessary for growth. Finally, essentialgrowth-promoting substances such as amino acids and vitamins can be usedin addition to the substances mentioned above. Said feed materials canbe added to the culture all at once or fed in appropriately duringcultivation.

The pH of the culture is controlled by the appropriate use of basiccompounds such as sodium hydroxide, potassium hydroxide or ammonia, oracid compounds such as phosphoric acid or sulfuric acid. Foaming can becontrolled using antifoams such as fatty acid polyglycol esters. Thestability of plasmids can be maintained by adding suitable selectivelyacting substances, e.g. antibiotics, to the medium. Aerobic conditionsare maintained by introducing oxygen or oxygen-containing gaseousmixtures, e.g. air, into the culture. The temperature of the culture isnormally 20° C. to 45° C. and preferably 25° C. to 40° C. The culture iscontinued until L-lysine formation has reached a maximum. This objectiveis normally achieved within 10 hours to 160 hours.

The concentration of L-lysine formed can be determined with the aid ofamino acid analyzers by means of ion exchange chromatography andpostcolumn reaction with ninhydrin detection, as described by Spackmannet al. (Analytical Chemistry 30, 1190 (1958)).

The following microorganisms have been deposited at the DeutscheSammlung für Mikroorganismen und Zellkulturen (German Collection ofMicroorganisms and Cell Cultures (DSMZ), Brunswick, Germany) under theterms of the Budapest Treaty:

-   -   Escherichia coli K-12 strain DH5α/pEC7pyc as DSM12870    -   Escherichia coli K-12 strain DH5α/pEC7dapBpyc as DSM12873    -   Escherichia coli K-12 strain DH5α/pEC7lysEdapBpyc as DSM12874    -   Corynebacterium glutamicum strain DSM5715/pJC23 as DSM12869    -   Corynebacterium glutamicum strain DSM5715aecD::dapA(MA16) as        DSM12867    -   Corynebacterium glutamicum strain DSM5715aecD::dapA(MC20) as        DSM12868    -   Corynebacterium glutamicum strain DM678 as DSM12866    -   Escherichia coli K-12 strain DH5α/pEC7lysEpyc as DSM 12872    -   Escherichia coli K-12 strain DH5α/pEC7dapBlysE as DSM 12875    -   Escherichia coli K-12 strain DH5α/pEC7lysE as DSM 12871

DETAILED DESCRIPTION OF THE INVENTION

The present invention is illustrated in greater detail below with theaid of Examples.

EXAMPLE 1 Preparation of the DNA Coding for the lysE Gene

Chromosomal DNA was isolated from the strain ATCC13032 by theconventional methods (Eikmanns et al., Microbiology 140: 1817-1828(1994)). The polymerase chain reaction (PCR) was used to amplify a DNAfragment carrying the lysE gene. The following primer oligonucleotideswere chosen for the PCR on the basis of the lysE gene sequence known forC. glutamicum (Vrljic et al., Molecular Microbiology 22(5), 815-826(1996)) (accession number X96471):

(SEQ ID NO: 7) LysBam1: 5′ CTC GAG AGC (GGA TCC) GCG CTG ACT CAC C 3′(SEQ ID NO: 8) LysBam2: 5′ GGA GAG TAC GGC (GGA TCC) ACC GTG ACC 3′

The primers shown were synthesized by MWG Biotech (Ebersberg, Germany)and the PCR was carried out by the standard PCR method of Innis et al.(PCR Protocols. A Guide to Methods and Applications, 1990, AcademicPress). The primers make it possible to amplify an approx. 1.1 kb DNAfragment carrying the lysE gene. The primers also contain the sequencefor the cleavage site of the restriction endonuclease BamHI, which isindicated by brackets in the nucleotide sequence shown above.

The amplified DNA fragment of approx. 1.1 kb, carrying the lysE gene,was identified by means of electrophoresis in 0.8% agarose gel, isolatedfrom the gel and purified with the QIAquick Gel Extraction Kit (cat. no.28704) from Qiagen (Hilden, Germany).

The fragment was then ligated by means of T4 DNA ligase from BoehringerMannheim (Mannheim, Germany) to vector pUC18 (Norrander et al., Gene(26) 101-106 (1983)). This was done by fully cleaving vector pUC18 withthe restriction endonuclease SmaI and treating it with alkalinephosphatase (Boehringer Mannheim, Mannheim, Germany). The ligationmixture was transformed to the E. coli strain DH5a (Hanahan, in: DNACloning. A Practical Approach, Vol. I, IRL-Press, Oxford, WashingtonD.C., USA). Plasmid-carrying cells were selected by plating thetransformation mixture on LB agar (Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.) which had been supplemented with 50 mg/l ofampicillin.

Plasmid DNA was isolated from a transformant and checked by treatmentwith the restriction enzyme BamHI followed by agarose gelelectrophoresis. The plasmid was called pUC18lysE.

EXAMPLE 2 Preparation of the dapB Gene

Chromosomal DNA was isolated from the Corynebacterium glutamicum strainATCC13032 as indicated in Example 1. The sequence of the dapB gene assuch from Corynebacterium glutamicum is known (accession number X67737).However, the published DNA sequence comprises only 56 bp upstream fromthe translation start, so the 5′ end upstream from the translation startwas additionally sequenced.

The sequencing was carried out with plasmid pJC25 (EP-B 0 435 132) usinga primer oligonucleotide which binds in the region of the known dapBsequence (accession number X67737). The sequence of the sequencingprimer used was:

(SEQ ID NO: 9) 5′ GAA CGC CAA CCT TGA TTC C 3′

The sequencing was carried out by the chain termination method describedby Sanger et al., Proc. Natl. Acad. Sci. USA, (74) 5463-5467 (1977). Thesequencing reaction was performed with the aid of the AutoReadSequencing Kit (Pharmacia, Freiburg). The electrophoretic analysis anddetection of the sequencing products were carried out with the A.L.F.DNA sequencer from Pharmacia (Freiburg, Germany).

The DNA sequence obtained was used to choose a second primer in order toobtain further sequence data upstream from the transcription start. Thefollowing primer was chosen for this purpose:

(SEQ ID NO: 10) 5′ CTT TGC CGC CGT TGG GTT C 3′

The sequencing reaction was carried out as described above. The novelsequence upstream from the dapB gene is shown as SEQ ID No. 1. Thesequence including the nucleotide sequence of the dapB gene is shown asSEQ ID No. 2.

The polymerase chain reaction was used to amplify the dapB gene. Forthis purpose, two primer oligonucleotides, chosen on the basis of theknown DNA sequence of the dapB gene, were synthesized by MWG Biotech:

(SEQ ID NO: 11) P-dap: 5′ (AAG CTT) AGG TTG TAG GCG TTG AGC 3′ (SEQ IDNO: 12) dapall: 5′ TTA ACT TGT TCG GCC ACA GC 3′

The 5′ primer (primer P-dap) contains a HindIII cleavage site which isindicated by brackets in the sequence shown above. The PCR was carriedout as in Example 1. An approx. 1.1 kb DNA fragment, which carries thedapB gene and contains a cleavage site for the restriction endonucleaseHindIII at each end, was amplified in this way. The PCR fragmentobtained was purified from 0.8% agarose gel (QIAquick Gel Extraction Kitfrom Qiagen, Hilden, Germany) and cloned into cloning vector pCR2.1TOPO(Invitrogen, Leek, The Netherlands) with the TOPO TA Cloning Kit(Invitrogen, Leek, The Netherlands, cat. no. K4550-01). The ligationmixture was transformed to the E. coli strain TOP10F′ from Invitrogen,the transformation mixture was plated on LB agar containing kanamycin(50 mg/l), IPTG (0.16 mM) and X-Gal (64 mg/l) and kanamycin-resistant,white colonies were isolated. Plasmid DNA was isolated from atransformant with the aid of the QIAprep Spin Miniprep Kit from Qiagenand checked by cleavage with the restriction enzyme HindIII followed byagarose gel electrophoresis. The DNA sequence of the amplified DNAfragment was checked by sequencing. The sequence of the PCR productmatches the sequence shown in SEQ ID No. 1. The plasmid obtained wascalled pCR2.1TOPOdapB.

EXAMPLE 3 Preparation of the DNA Coding for the pyc Gene

The Corynebacterium glutamicum strain ATCC13032 was used as the donorfor the chromosomal DNA. Chromosomal DNA was isolated from the strainATCC13032 as described in Example 1. The polymerase chain reaction wasused to amplify a DNA fragment carrying the pyc gene. The followingprimer oligonucleotides were chosen for the PCR on the basis of the pycgene sequence known for C. glutamicum (Peters-Wendisch et al.,Microbiology 144, 915-927 (1998)) (accession number Y09548):

(SEQ ID NO: 13) 5-PYC-IN: 5′ GC(T CTA GA)A GTG TCG CAA CCG TGC TTG A 3′(SEQ ID NO: 14) 3-PYC-IN: 5′ GC(T CTA GA)T TGA GCC TTG GTC TCC ATC T 3′

The primers shown were synthesized by MWG Biotech and the PCR reactionwas carried out by the standard PCR method of Innis et al. (PCRProtocols. A Guide to Methods and Applications, 1990, Academic Press).The primers make it possible to amplify an approx. 3.8 kb DNA fragmentcarrying the pyc gene. The primers also contain the sequence for acleavage site of the restriction endonuclease XbaI, which is indicatedby brackets in the nucleotide sequence shown above.

The amplified DNA fragment of approx. 3.8 kb, carrying the pyc gene, wasidentified by gel electrophoresis in 0.8% agarose gel, isolated from thegel and purified by the conventional methods (QIAquick Gel ExtractionKit, Qiagen, Hilden).

The fragment was then ligated to vector pCRII-TOPO by means of the DualPromotor Topo TA Cloning Kit (Invitrogen, Leek, The Netherlands, cat.number K4600-01). The ligation mixture was transformed to the E. colistrain TOP10 (Invitrogen, Leek, The Netherlands). Plasmid-carrying cellswere selected by plating the transformation mixture on LB agarcontaining kanamycin (50 mg/l) and X-Gal (64 mg/l).

After isolation of the DNA, the plasmid obtained was checked by means ofrestriction cleavage and identified in agarose gel. The plasmid wascalled pCRII-TOPOpyc and the DNA sequence of the cloned insert wassequenced for control purposes. As the determined sequence of the pycinsert in pCRII-TOPOpyc matches the sequence of the gene library entry,this plasmid was used subsequently.

EXAMPLE 4 Cloning of the dapB Gene Into Vector pEC7

An approx. 1.1 kb DNA fragment carrying the dapB gene was isolated fromplasmid pCR2.1TOPOdapB (from Example 2). For this purpose, plasmidpCR2.1TOPOdapB was fully digested with the restriction enzyme HindIIIand the approx. 1.1 kb DNA fragment carrying the dapB gene was isolated.

The dapB fragment was inserted into vector pEC7. Vector pEC7 is based onE. coli—C. glutamicum shuttle vector pEC5 (Eikmanns et al., 102: 93-98(1991)). The BamHI cleavage site not located in the polylinker wasremoved from plasmid pEC5 in the following manner: Plasmid pEC5 waspartially cleaved with the restriction enzyme BamHI. The approx. 7.2 kbDNA fragment was isolated from the agarose gel and the protruding endswere filled in with Klenow polymerase (Boehringer Mannheim). Theresulting DNA fragment was ligated (T4 ligase, Boehringer Mannheim). Theligation mixture was transformed to the E. coli strain DH5α andkanamycin-resistant colonies were isolated on LB agar containingkanamycin (50 mg/l). Plasmid DNA was isolated from a transformant(QIAprep Spin Miniprep Kit from Qiagen) and checked by restrictioncleavage with the restriction enzymes BamHI and PstI. The resultingplasmid was called pEC6.

Plasmid pEC6 was fully cleaved with the restriction enzyme XhoI. A DNAfragment carrying the trp terminator was ligated to vector DNA fragment(T4 ligase, Boehringer Mannheim). The ligation mixture was transformedto the E. coli strain DH5α and kanamycin-resistant colonies wereisolated on LB agar containing kanamycin (50 mg/l). Plasmid DNA wasisolated from a transformant (QIAprep Spin Miniprep Kit from Qiagen) andchecked by restriction cleavage with the restriction enzymes BamHI andXhoI. The resulting plasmid was called pEC7.

The dapB-carrying DNA fragment obtained was ligated to vector pEC7 (T4DNA ligase, Boehringer Mannheim), which had also been fully digestedwith the restriction enzyme HindIII and treated with alkalinephosphatase (Boehringer Mannheim). The ligation mixture was transformedto the E. coli strain DH5α and kanamycin-resistant colonies wereisolated on LB agar containing kanamycin (50 mg/l). Plasmid DNA wasisolated from a transformant (QIAprep Spin Miniprep Kit from Qiagen) andchecked by restriction cleavage with the restriction enzyme HindIII. Theresulting plasmid was called pEC7dapB (FIG. 1). The Escherichia coli lstrain obtained was called DH5α/pEC7dapB.

EXAMPLE 5 Cloning of the lysE Gene Into Vector pEC7

Plasmid pUC18lysEneu described in Example 1 was fully digested with therestriction enzyme BamHI and the 1.1 kb BamHI fragment carrying the lysEgene was isolated as in Example 1. Vector pEC7 was likewise fullycleaved with the restriction enzyme BamHI and treated with alkalinephosphatase. The BamHI vector fragment and the BamHI lysE fragment wereligated (Rapid DNA Ligation Kit, Boehringer Mannheim) and transformed tothe E. coli strain DH5α. Plasmid-carrying transformants were selected onLB agar containing chloramphenicol (10 mg/l). Plasmid DNA was isolated(QIAprep Spin Miniprep Kit, Qiagen) and checked by restriction cleavagewith the enzyme BamHI. The resulting plasmid was called pEC7lysE (FIG.2). The strain obtained by transformation of plasmid pEC7lysE to the E.coli strain DH5α was called DH5α/pEC7lysE.

EXAMPLE 6 Cloning of the pyc Gene Into Vector pEC7

The 3.8 kb DNA fragment carrying the pyc gene from C. glutamicumATCC13032 was obtained from plasmid pCRII-TOPOpyc (from Example 3) bycleavage with the restriction enzyme XbaI. The 3.8 kb DNA fragment wasidentified by gel electrophoresis, isolated from the gel and purified bythe conventional methods and the protruding ends were filled in withKlenow polymerase. Vector pEC7 was likewise fully cleaved with therestriction enzyme SmaI and treated with alkaline phosphatase. The SmaIvector fragment and the XbaI pyc fragment treated with Klenow polymerasewere ligated (T4 ligase, Boehringer Mannheim) and transformed to the E.coli strain DH5α. Plasmid-carrying transformants were selected on LBagar containing chloramphenicol (10 mg/l). Plasmid DNA was isolated(QIAprep Spin Miniprep Kit, Qiagen, Hilden, Germany) and checked byrestriction cleavage with the restriction enzyme SalI. The resultingplasmid was called pEC7pyc (FIG. 3). The E. coli strain obtained bytransformation of plasmid pEC7pyc to the E. coli strain DH5α was calledDH5α/pEC7pyc.

EXAMPLE 7 Preparation of a Plasmid Containing lysE and dapB Genes

The dapB gene was isolated as a HindIII fragment from plasmidpCR2.1TOPOdapB containing the dapB gene from C. glutamicum ATCC13032. Todo this, the plasmid was fully digested with the restriction enzymeHindIII and the dapB-carrying DNA fragment was isolated from 0.8%agarose gel (QIAquick Gel Extraction Kit, Qiagen).

Vector pEC7lysE was also fully digested with the restriction enzymeHindIII and treated with alkaline phosphatase. The 1.1 kb fragmentcontaining dapB was ligated to the resulting linear vector fragment (T4ligase, Boehringer Mannheim) and the ligation mixture was transformed tothe E. coli strain DH5α. Plasmid-carrying transformants were selected onLB agar containing chloramphenicol (10 mg/l). Plasmid DNA was isolated(QIAprep Spin Miniprep Kit, Qiagen, Hilden, Germany) and checked byrestriction cleavage with the restriction enzyme HindIII.

The resulting plasmid was called pEC7lysEdapB. This plasmid is capableof autonomous replication in Escherichia coli and in Corynebacterium andconfers resistance to the antibiotic chloramphenicol on its host.

Plasmid pEC7lysEdapB simultaneously contains the dapB gene, which codesfor dihydrodipicolinate reductase, and the lysE gene, which codes forthe lysine exporter.

The strain obtained by the transformation of E. coli DH5α withpEC7lysEdapB was called DH5α/pEC7lysEdapB.

EXAMPLE 8 Preparation of a Plasmid Simultaneously Containing dapB andpyc Genes

The plasmid carrying the pyc gene which codes for the pyruvatecarboxylase from Corynebacterium glutamicum ATCC13032 was fully cleavedwith the restriction enzyme XbaI and the protruding ends were filled inwith Klenow polymerase as described in Example 6, making it possible toisolate the 3.8 kb DNA fragment containing the gene for pyruvatecarboxylase.

Plasmid pEC7dapB (from Example 4) was also fully cleaved with therestriction enzyme SmaI and the ends were treated with alkalinephosphatase. The resulting linear vector fragment was ligated to the 3.8kb DNA fragment containing the pyc gene using T4 DNA ligase (BoehringerMannheim, Mannheim, Germany). This produced a plasmid containing boththe dapB gene and the pyc gene from corynebacteria. Plasmid-carryingtransformants were selected on LB agar containing chloramphenicol (10mg/l). Plasmid DNA was isolated (QIAprep Spin Miniprep Kit, Qiagen,Hilden, Germany) and verified by restriction cleavage with therestriction enzyme SalI. The plasmid is shown in FIG. 4 and was calledpEC7dapBpyc. The E. coli strain obtained by transformation of plasmidpEC7dapBpyc to the E. coli strain DH5α was called DH5α/pEC7dapBpyc.

EXAMPLE 9 Preparation of a Plasmid Containing Sequences SimultaneouslyCoding for the lysE, dapB and pyc Genes

Plasmid pCRII-TOPOpyc (from Example 3), which carries the pyc genecoding for the pyruvate carboxylase from Corynebacterium glutamicumATCC13032, was fully cleaved with the restriction enzyme XbaI andtreated with Klenow polymerase as described in Example 6, making itpossible to isolate the 3.8 kb DNA fragment containing the gene forpyruvate carboxylase.

Plasmid pEC7dapBlysE was also fully cleaved with the restriction enzymeSmaI and the ends were treated with alkaline phosphatase. The resultinglinear vector fragment was ligated to the 3.8 kb DNA fragment containingthe pyc gene using T4 DNA ligase (Boehringer Mannheim). This produces aplasmid containing the lysE gene and dapB gene and the pyc gene fromCorynebacterium glutamicum. Plasmid-carrying transformants were selectedon LB agar containing chloramphenicol (10 mg/l). Plasmid DNA wasisolated (QIAprep Spin Miniprep Kit, Qiagen, Hilden, Germany) andverified by restriction cleavage with the restriction enzyme SmaI. Theplasmid is shown in FIG. 5 and was called pEC7lysEdapBpyc. The E. colistrain obtained by transformation of plasmid pEC7dapBlysEpyc to the E.coli strain DH5α was called DH5α/pEC7dapBlysEpyc.

EXAMPLE 10

Transformation of the strain MH20-22B with plasmids pJC23 and pJC33

Plasmid pJC1 is a plasmid capable of replication in Escherichia coli andCorynebacterium glutamicum (Cremer et al., Molecular and GeneralGenetics 220: 478-480 (1990)). Plasmid pJC33 (Cremer et al., Applied andEnvironmental Microbiology 57(6), 1746-1752 (1991)), which carries thelysC (Fbr) gene from the C. glutamicum strain MH20-22B, is derivedtherefrom.

Plasmid pJC23 is also based on vector pJC1 and carries the dapA genefrom C. glutamicum ATCC13032 (Cremer et al., Molecular and GeneralGenetics 220: 478-480 (1990)) (EP-B 0 435 132). Plasmids pJC1, pJC33 andpJC23 were introduced into the strain MH20-22B by the electroporationmethod (Haynes and Britz, FEMS Microbiology Letters (61) 329-334(1989)). The C. glutamicum strain MH20-22B is an AEC-resistant lysineproducer deposited under the number DSM5715.

The transformants obtained by means of electroporation were isolated onselection agar (LBHIS agar (18.5 g/l of brain-heart infusion broth, 0.5M sorbitol, 5 g/l of bacto tryptone, 2.5 g/l of bacto yeast extract, 5g/l of NaCl, 18 g/l of bacto agar)) containing 15 mg/l of kanamycin.Plasmid DNA was isolated by the conventional methods (Peters-Wendisch etal., Microbiology 144, 915-927 (1998)), cleaved with suitablerestriction endonucleases and checked. The strains obtained were calledMH20-22B/pJC1, MH20-22B/pJC33 and MH20-22B/pJC23.

EXAMPLE 11 Transformation with Plasmids pEC7pyc, pEC7dapBpyc andpEC7lysEdapBpyc

The strains prepared in Example 10 were subsequently provided with asecond plasmid.

The following plasmids were introduced by the electroporation methodinto the strains MH20-22B/pJC1, MH20-22B/pJC33 and MH20-22B/pJC23described:

pEC7pyc (cf. Example 6) pEC7dapBpyc (cf. Example 8) pEC7lysEdapBpyc (cf.Example 9)

The transformed bacteria are selected on the basis of the antibioticresistance of the plasmids they contain. The transformants obtained bymeans of electroporation were isolated on selection agar (LBHIS agarcontaining 15 mg/l of kanamycin and 7.5 mg/l of chloramphenicol).Plasmid DNA was isolated, cleaved with suitable restrictionendonucleases and checked.

The strains obtained are listed below:

-   DSM5715/pJC1/pEC7pyc-   DSM5715/pJC33/pEC7pyc-   DSM5715/pJC23/pEC7pyc-   DSM5715/pJC23/pEC7dapBpyc-   DSM5715/pJC23/pEC7lysEdapBpyc

EXAMPLE 12 Preparation of L-lysine

The various C. glutamicum strains obtained in Examples 10 and 11 werecultivated in a nutrient medium suitable for lysine production and thelysine content of the culture supernatant was determined.

This was done by first incubating the various strains on agar plateswith the appropriate antibiotics (brain-heart agar containing kanamycin(25 mg/1) and chloramphenicol (10 mg/l)) for 24 hours at 33° C. Theseagar plate cultures were used to inoculate a preculture (10 ml of mediumin a 100 ml conical flask). Complete medium CgIII was used as thepreculture medium. Kanamycin (25 mg/l) and chloramphenicol (10 mg/l)were added. The preculture was incubated for 24 hours at 33° C. on ashaker at 250 rpm. This preculture was used to inoculate a main cultureto give an initial OD (660 nm) of 0.2. Medium MM was used for the mainculture.

Medium MM CSL 5 g/l MOPS 20 g/l Glucose 50 g/l (autoclave separately)Salts: (NH₄)₂SO₄ 25 g/l KH₂PO₄ 0.1 g/l MgSO₄*7H₂O 1.0 g/l CaCl₂*2H₂O 10mg/l FeSO₄*7H₂O 10 mg/l MnSO₄*H₂O 5.0 mg/l Biotin 0.3 mg/l(sterile-filtered) Thiamine*HCl 0.2 mg/l (sterile-filtered) CaCO₃ 25 g/lAbbreviations: CSL: corn steep liquor MOPS: morpholinopropanesulfonicacid

CSL, MOPS and the salt solution are adjusted to pH 7 with aqueousammonia and autoclaved. The sterile substrate and vitamin solutions andthe dry-autoclaved CaCO₃ are then added.

Cultivation is carried out in a volume of 10 ml in a 100 ml conicalflask with baffles. Kanamycin (25 mg/l) and chloramphenicol (10 mg/l)were added. Cultivation proceeded at 33° C. and 80% atmospherichumidity.

After 72 hours the OD was measured at a wavelength of 660 nm. The amountof lysine formed was determined with an amino acid analyzer fromEppendorf BioTronik (Hamburg, Germany) by means of ion exchangechromatography and postcolumn derivation with ninhydrin detection. Theglucose content was determined with a sugar analyzer from SkalarAnalytik GmbH (Erkelenz, Germany).

The experimental results are shown in Table 1.

TABLE 1 OD (660 Lysine-HCl Strain Gene nm) g/l DSM5715/pJC1/pEC7pyc pyc9.3 11.3 DSM5715/pJC33/pEC7pyc pyc, lysC(Fbr) 9.2 11.9DSM5715/pJC23/pEC7pyc pyc, dapA 9.3 14.4 DSM5715/pJC23/pEC7dapBpyc pyc,dapA, dapB 9.3 16.9 DSM5715/pJC23/ pyc, dapA, dapB, 9.1 17.6pEC7lysEdapBpyc lysE

EXAMPLE 13 Cloning of the aecD Gene into Vector pUC18

Plasmid pSIR21 (Rossol, Thesis, University of Bielefeld 1992) was fullycleaved with the enzymes BglII and EcoRV and the 1.4 kb DNA fragmentcontaining the aecD gene (accession number M89931) (Rossol and Pühler,Journal of Bacteriology 174 (9), 2968-2977 (1992)) from C. glutamicumATCC13032 was isolated. The isolated DNA fragment was ligated to plasmidpUC18 (which had been fully digested with the enzymes BamHI and SmaI)using T4 DNA ligase, as described in Sambrook et al. (Molecular Cloning:A Laboratory Manual (1989), Cold Spring Harbor Laboratory Press). Theligation mixture was transformed to the E. coli strain DH5α. Thetransformants were selected on brain-heart agar plates containing 100mg/l of ampicillin. Plasmid DNA was isolated from one colony. Theplasmid obtained was called pUC18::aecD.

EXAMPLE 14 Cloning of the dapA Gene into Plasmid pSP72

A dapA gene fragment is isolated from plasmid pJC20 (Cremer, J., Thesis1989, University of Düsseldorf) as an SphI-BamHI fragment. Vector pSP72(Promega Corporation, USA) was fully cleaved with the enzymes SphI andBamHI and treated with alkaline phosphatase. The dapA-carrying fragmentwas ligated to this vector using T4 DNA ligase. The DNA was thentransformed to the E. coli strain XL1 Blue (Bullock, Fernandez andShort, BioTechniques (5), 376-379 (1987)). The transformants wereselected on LB medium containing 100 mg/l of ampicillin. Plasmid DNA wasisolated from one transformant and called pSP72::dapA.

EXAMPLE 15 Mutagenesis of the dapA Promoter and Preparation of PlasmidspSP72::dapA (MC20) and pSP72::dapA (MA16)

The Quickchange site directed mutagenesis kit from Stratagene was usedfor the mutagenesis of the promoter region. The following primers wereconstructed with the aid of said dapA sequence and used for themutagenesis:

For the preparation of pSP72::dapA (MC20)

(SEQ ID NO: 15) Primer dap1 for MC20 CCA AAT GAG AGA TGG TAA CCT TGA ACTCTA TGA GCA (SEQ ID NO: 16) Primer dap2 for MC20 GTG CTC ATA GAG TTC AAGGTT ACC ATC TTC CCT CAT TTG GFor the preparation of pSP72::dapA(MA16)

(SEQ ID NO: 17) Primer dap3 for MA16 CCA AAT GAG GGA AGA AGG TAT AAT TGAACT CTA TGA GCA (SEQ ID NO: 18) Primer dap4 for MA16 GTG CTC ATA GAG TTCAAT TAT ACC TTC TTC CCT CAT TTG G

The PCR was carried out as indicated by the manufacturer of theQuickchange site directed mutagenesis kit (Stratagene) using plasmidpSP72::dapA (from Example 14) as the template.

The mutagenesis mixtures were transformed to the E. coli strain XL1Blue. The transformants were selected on LB medium containing 100 mg/lof carbenicillin. Plasmid DNA was isolated from one transformant and theloss of the BstEII cleavage site was controlled by BstEII digestion.Plasmids no longer carrying a BstEII cleavage site exhibited the desiredmutation.

The plasmids obtained were transformed to the dapA-defective E. colimutant RDA8. The transformation mixtures were plated on LB containing100 mg/l of carbenicillin in order to test the complementation of thedapA mutation. DNA was isolated from one transformant in each case andthe plasmids obtained were called pSP72::dapA(MC20) andpSP72::dapA(MA16). The plasmids were sequenced by the chain terminationmethod described in Sanger et al., Proceedings of the National Academyof Sciences of the USA (74), 5463-5467 (1977), using the reverse anduniversal sequencing primers. The sequencing reaction was performed withthe aid of the AutoRead Sequencing Kit (Pharmacia, Freiburg). Theelectrophoretic analysis and detection of the sequencing products werecarried out with the A.L.F. DNA sequencer (Pharmacia, Freiburg,Germany).

EXAMPLE 16 Preparation of Plasmids pK19mobsacBaecD::dapA (MC20) andpK19mobsacBaecD::dapA (MA16) (Recloning of the Mutagenized Fragments)

Plasmids pSP72::dapA(MC20) and pSP72::dapA (MA16) (from Example 15) werefully cleaved with the restriction enzymes PvuII and SmaI. The 1450 bpPvuII-SmaI fragments carrying the dapA gene with the mutated MC20 orMA16 promoter were ligated to StuI-cleaved vector pUC18::aecD (fromExample 13) using T4 DNA ligase. The ligation mixture was transformed tothe E. coli strain DH5α. The transformants were selected on LB mediumcontaining 100 mg/l of ampicillin. Plasmid DNA was isolated from onetransformant in each case to give plasmids pUC18aecD::dapA (MC20) andpUC18aecD::dapA (MA16).

Plasmids pUC18aecD::dapA (MC20) and pUC18aecD::dapA (MA16) werepartially cleaved with the restriction enzyme EcoRI and fully cleavedwith the enzyme SalI to give the 3.0 kb fragment carrying aecD::dapA(MA16) or aecD::dapA (MC20). The fragment was ligated to vectorpK19mobsacB (which had been cleaved and treated with alkalinephosphatase) (Schäfer et al., Gene (145), 69-73 (1994)) using T4 DNAligase. The ligation mixture was transformed to the E. coli strain DH5(Hanahan (1985), in: DNA Cloning. A Practical Approach, Vol. I,IRL-Press, Oxford, Washington D.C., USA). The transformants wereselected on LB medium containing 50 mg/l of kanamycin. Plasmid DNA wasisolated from one transformant in each case to give plasmidspK19mobsacBaecD::dapA (MC20) and pK19mobsacBaecD::dapA (MA16).

The plasmid DNA was transformed to the E. coli strain S17-1 (Simon,Priefer and Pühler, Bio/Technology (1), 784-791 (1983)). Thetransformants were selected on LB medium containing 50 mg/l ofkanamycin. Plasmid DNA was isolated from one transformant in each caseand checked. The strains obtained were calledS17-1/pK19mobsacBaecD::dapA (MC20) and S17-1/pK19mobsacBaecD::dapA(MA16).

EXAMPLE 17 Preparation of the C. glutamicum Strains DSM5715aecD::dapA(MC20) and DSM5715aecD::dapA (MA16)

Plasmids pK19mobsacBaecD::dapA (MC20) and pK19mobsacBaecD::dapA (MA16)were transferred from S17-1/pK19mobsacBaecD::dapA (MC20) andS17-1/pK19mobsacBaecD::dapA (MA16) (from Example 16) to the C.glutamicum strain DSM5715 by the conjugation method (Schäfer et al.,Journal of Bacteriology (172), 1663-1666 (1990)). For selection of thetransconjugants, the conjugation mixtures were plated on brain-heartmedium containing nalidixic acid and kanamycin. The transconjugantsobtained were incubated overnight in 10 ml of brain-heart medium.Aliquots were then plated on plates containing sucrose (brain-heart agarcontaining 10% of sucrose) in order to select for loss of sucrosesensitivity. Sucrose-resistant clones were isolated and checked again onagar plates containing chloramphenicol and kanamycin (brain-heart mediumcontaining 15 mg/l of kanamycin and brain-heart medium containing 10mg/l of chloramphenicol).

Colonies exhibiting the following phenotype were isolated:

-   sucrose resistant-   kanamycin sensitive-   chloramphenicol sensitive

The insertion of the dapA gene fragment into the aecD gene was checkedby the Southern blot method (Sambrook et al., Molecular Cloning: ALaboratory Manual (1989), Cold Spring Harbor Laboratory Press).

EXAMPLE 18 Preparation of the C. glutamicum Strains DSM5715aecD::dapA(MC20)/pEC7pyc, DSM5715aecD::dapA (MA16)/pEC7pyc, DSM5715aecD::dapA(MC20)/pEC7, DSM5715aecD::dapA (MA16)/pEC7 and DSM5715/pEC7

As described in Example 6, the pyc gene is present in vector pEC7pyc.This plasmid pEC7pyc and plasmid pEC7 were introduced into the strainsDSM5715aecD::dapA (MC20), DSM5715aecD::dapA (MA16) and DSM5715 (fromExample 17) by means of electroporation (Haynes 1989, FEMS MicrobiologyLetters 61, 329-334) to give C. glutamicum DSM5715aecD::dapA(MC20)/pEC7pyc, DSM5715aecD::dapA (MA16)/pEC7pyc, DSM5715aecD::dapA(MC20)/pEC7, DSM5715aecD::dapA (MA16)/pEC7 and DSM5715/pEC7. Thetransformants were selected on brain-heart agar containing 25 mg/l ofkanamycin. Plasmid DNA was isolated from one transformant in each caseand checked.

The following strains were obtained in this way:

-   DSM5715aecD::dapA (MC20)/pEC7pyc,-   DSM5715aecD::dapA (MA16)/pEC7pyc,-   DSM5715aecD::dapA (MC20)/pEC7,-   DSM5715aecD::dapA (MA16)/pEC7 and-   DSM5715/pEC7.

EXAMPLE 19 Preparation of Lysine with the Strains Prepared in Example 18

After precultivation in CgIII medium (Kase & Nakayama, Agricultural andBiological Chemistry 36 (9), 1611-1621 (1972)), the strainsDSM5715aecD::dapA (MC20)/pEC7pyc, DSM5715aecD::dapA (MA16)/pEC7pyc,DSM5715aecD::dapA (MC20)/pEC7, DSM5715aecD::dapA (MA16)/pEC7 andDSM5715/pEC7 were cultivated in MM production medium as described inExample 12. After incubation for 48 hours, the optical density at 660 nmand the concentration of L-lysine formed were determined.

The experimental results are shown in Table 2.

TABLE 2 OD Lysine-HCl Strain (660 nm) g/lDSM5715aecD::dapA(MC20)/pEC7pyc 14.15 13.3DSM5715aecD::dapA(MA16)/pEC7pyc 13.6 13.4 DSM5715/pEC7 13.3 11.5DSM5715aecD::dapA(MA16)/pEC7 13.3 12.9 DSM5715aecD::dapA(MC20)/pEC7 14.012.8

EXAMPLE 20 Transformation of Strain DM678 with Plasmids pEC7 andpEC7lysEdapBpyc

The lysine producing C. glutamicum strain DM678 was derived fromATCC13032 by several rounds of mutagenesis and screening. It has apartial requirement for L-threonine and its growth is sensitive towardsL-methionine. Strain DM678 is deposited as DSM12866 at the DeutscheSammlung für Mikroorganismen und Zellkulturen (DSMZ, Braunschweig,Deutschland).

Plasmids pEC7 (from Example 4) and pEC7lysEdapBpyc (from Example 9) wereintroduced into the strain DM678 by the electroporation method (Haynesand Britz, FEMS Microbiology Letters (61) 329-334 (1989)).

The transformants obtained by means of electroporation were isolated onselection agar (LBHIS agar (18.5 g/l of brain-heart infusion broth, 0.5M sorbitol, 5 g/l of bacto tryptone, 2.5 g/l of bacto yeast extract, 5g/l of NaCl, 18 g/l of bacto agar)) containing 7.5 mg/l ofchloramphenicol. Plasmid DNA was isolated by the conventional methods(Peters-Wendisch et al., Microbiology 144, 915-927 (1998)), cleaved withsuitable restriction endonucleases and checked by agarosegelelectrophoresis. The strains obtained were called DM678/pEC7 andDM678/pEC7lysEdapBpyc.

EXAMPLE 21 Preparation of L-lysine

The C. glutamicum strains obtained in Examples 20 were cultivated in anutrient medium suitable for lysine production and the lysine content ofthe culture supernatant was determined.

This was done by first incubating the strains on agar plates(brain-heart) with chloramphenicol (10 mg/l) for 24 hours at 33° C.These agar plate cultures were used to inoculate a preculture (10 ml ofmedium in a 100 ml conical flask). Complete medium CgIII which had beensupplemented with 10 mg/l of chloramphenicol was used as the preculturemedium. The preculture was incubated for 24 hours at 33° C. on a shakerat 250 rpm. This preculture was used to inoculate a main culture to givean initial OD (660 nm) of 0.1. Medium MM2 was used for the main culture.

Medium MM2: CSL 25 g/l MOPS 20 g/l Glucose 80 g/l (autoclave separately)Salts: (NH₄)₂SO₄ 25 g/l KH₂PO₄ 0.1 g/l MgSO₄*7H₂O 0.25 g/l CaCl₂*2H₂O 10mg/l FeSO₄*7H₂O 10 mg/l MnSO₄*H₂O 10 mg/l Biotin 0.3 mg/l(sterile-filtered) Thiamine*HCl 0.2 mg/l (sterile-filtered) CaCO₃ 25 g/l(autoclave separately) Abbreviations: CSL: corn steep liquor MOPS:morpholinopropanesulfonic acid

CSL, MOPS and the salt solution are adjusted to pH 7 with aqueousammonia and autoclaved. The sterile substrate and vitamin solutions, theChloramphenicol (10 mg/l) and the dry-autoclaved CaCO₃ are then added.

Cultivation is carried out in a volume of 10 ml in a 100 ml conicalflask with baffles. Incubation proceeded at 33° C. and 80% atmospherichumidity.

After 48 hours the OD was measured at a wavelength of 660 nm. The amountof lysine formed was determined with an amino acid analyzer fromEppendorf BioTronik (Hamburg, Germany) by means of ion exchangechromatography and postcolumn derivation with ninhydrin detection. Theglucose content was determined with a sugar analyzer from SkalarAnalytik GmbH (Erkelenz, Germany).

The experimental results are shown in Table 3.

TABLE 3 OD Lysine-HCl Strain Gene (660 nm) g/l DM678/pEC7 25.6 16.7DM678/ pyc, dapB, lysE 23.3 17.7 pEC7lysEdapBpyc

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the pEC7dapB plasmid (cf. Example 4)

FIG. 2 illustrates the pEC7lysE plasmid (cf. Example 5)

FIG. 3 illustrates the pEC7pyc plasmid (cf. Example 6)

FIG. 4 illustrates the pEC7dapBpyc plasmid (cf. Example 8)

FIG. 5 illustrates the pEC7lysEdapBpyc plasmid (cf. Example 9).

The abbreviations used in the Figures are defined as follows:

Cm: chloramphenicol resistance gene dapB: dapB gene from C. glutamicumlysE: lysE gene from C. glutamicum pyc: pyc gene from C. glutamicumOriE: plasmid-coded origin of replication of E. coli pBL: DNA fragmentof plasmid pBL1 EcoRI: cleavage site of the restriction enzyme EcoRIEcoRV: cleavage site of the restriction enzyme EcoRV HincII: cleavagesite of the restriction enzyme HincII HindIII: cleavage site of therestriction enzyme HindIII KpnI: cleavage site of the restriction enzymeKpnI SalI: cleavage site of the restriction enzyme SalI SmaI: cleavagesite of the restriction enzyme SmaI SphI: cleavage site of therestriction enzyme SphI PvuII: cleavage site of the restriction enzymePvuII BamHI: cleavage site of the restriction enzyme BamHI

1. An L-Lysine-producing bacterium of the species Corynebacteriumglutamicum comprising: a) an overexpressed wild type pyc gene ofCorynebacterium glutamicum encoding pyruvate carboxylase, whereinoverexpression of said pyc gene is achieved by increasing the copynumber of said pyc gene, and b) an overexpressed wild type dapA gene ofCorynebacterium glutamicum encoding dihydrodipicolinate synthase,wherein overexpression of said dapA gene is achieved by using a dapApromoter selected from the group consisting of: the dapA promotercomprising the MC20 mutation as set forth in SEQ ID NO: 5 and the dapApromoter comprising the MA16 mutation as set forth in SEQ ID NO: 6, andwhereby said overexpression of said wild type pyc gene ofCorynebacterium glutamicum or said wild type dapA gene ofCorynebacterium glutamicum results in pyruvate carboxylase activity ordihydrodipicolinate synthase activity above the level of that found in awild type Corynebacterium glutamicum.
 2. An Escherichia coli K-12 strainDH5α/pEC7lysEpyc, deposited as DSM12872.
 3. An isolated DNA comprisingthe nucleotide sequence shown in SEQ ID NO:
 5. 4. An isolated DNAcomprising the nucleotide sequence shown in SEQ ID NO:
 6. 5. Thebacterium of claim 1 further comprising an overexpressed lysC gene ofCorynebacterium glutamicum encoding aspartate kinase, wherein said geneis expressed at a level that is higher than its expression level in wildtype Corynebacterium glutamicum by increasing the copy number of saidgene.
 6. An L-Lysine-producing bacterium of the species Corynebacteriumglutamicum comprising: a) an overexpressed wild type pyc gene ofCorynebacterium glutamicum coding pyruvate carboxylase, whereinoverexpression of said pyc gene is achieved by increasing the copynumber of said pyc gene, b) an overexpressed wild type dapA gene ofCorynebacterium glutamicum encoding dihydrodipicolinate synthase,wherein overexpression of said dapA gene is achieved by using a dapApromoter selected from the group consisting of: the dapA promotercomprising the MC20 mutation as set forth in SEQ ID NO: 5 and the dapApromoter comprising the MA16 mutation as set forth in SEQ ID NO: 6, andc) an overexpressed wild type lysE gene of Corynebacterium glutamicumencoding a lysine export carrier, wherein overexpression of said lysEgene is achieved by increasing the copy number of said lysE gene, andwherein the overexpressed genes are expressed at levels that are higherthan their respective expression levels in wild type Corynebacteriumglutamicum.
 7. The bacterium of claim 6 further comprising anoverexpressed lysC gene of Corynebacterium glutamicum encoding aspartatekinase wherein said gene is expressed at a level that is higher than itsexpression level in wild type Corynebacterium glutamicum andoverexpression of said gene is achieved by increasing the copy number ofsaid gene.